GLUTAMATE RECEPTOR-DRIVEN ACTIVATION OF TRANSCRIPTION FACTORS IN PRIMARY NEURONAL CULTURES

We have used primary neuronal cultures prepared from fetal cerebral he mispheres to investigate the effects of different glutamate receptor a gonists and antagonists on the expression of transcription factor enco ding genes, such as c-fos, fosB, c-jun, junB, junD, c-myc, and zif/268 . The addition of glutamate (100 mu M) to the culture medium rapidly a ctivated c-fos, fosB, c-jun, junB and zif/ 268 gene expression, reachi ng the maximal level at 30-60 minutes for zif/268 and at 60 minutes fo r the other genes. The onset of fosB mRNA accumulation was slightly de layed in comparison to the other genes. No clear induction was found f or junD and c-myc. Different glutamate receptor agonists, such as NMDA , kainate, quisqualate, trans-(+/-)-1-aminocyclopentane-1,3-dicarboxyl ic acid (t-ACPD) and ha-amino-3-hydroxy-5-methyl-4-isoxazole-propionat e (AMPA) were able to increase crease c-fos, c-jun, and zif-268 mRNA l evels with rapid and transient kinetics similar to those observed afte r glutamate treatment. Similar results were obtained for junB and fosB after kainate and quisqualate stimulation. Pretreatment with MK-801, a non competitive NMDA antagonist, produced an almost complete inhibit ion of glutamate-driven expression of transcription factor genes, thus suggesting that NMDA receptor plays a major role in glutamate induced -gene expression. On the contrary the kainate/AMPA receptor antagonist , DNQX, did not influence glutamate induced-gene expression. Under the conditions used in the present study, NMDA was effective in inducing the simultaneous activation of several IEGs even when added to the cul ture medium containing millimolar concentration of magnesium. When exp eriments were performed in Krebs solution, NMDA was effective in stimu lating zif/268 and c-fos mRNAs only in the absence of Mg2+, while glut amate activated c-fos and zif/268 both in the presence and absence of magnesium ions. As expected, NMDA effect was fully inhibited by MK-801 . The level of AP-1 DNA binding activity, as measured by electrophoret ic mobility shift assay, increased after addition of glutamate and NMD A to cultured neurons and such increase was antagonized by the pretrea tment with MK-801.